Almost 60% of the 40 million HIV-1 infected individuals worldwide are women, and most new HIV-1 infections are contracted by heterosexual contact. Mucosal HIV transmission is therefore the most important target for preventative strategies. To design vaccines or topical microbicides that interfere with viral transmission, it is critical to have an in-depth understanding of the initial infection events in the mucosa. However, the exact pathways whereby HIV enters and propagates in the mucosa remain ill defined. In particular, data are lacking that clarify how HIV interacts with Langerhans and CD4+ T cells that reside in the outer epithelial layer of the human vagina or ectocervix. These cells are presumably the first susceptible targets for HIV, and a successful blockade of their infection could be extremely effective in aborting viral transmission. To address these issues, we developed a novel ex vivo organ culture model of the human vaginal mucosa. This model allows us to perform detailed in situ investigations of the productive and non- productive pathways for entry of HIV into intraepithelial Langerhans and T cells. In Aim 1, we will use our model to characterize the initial HIV-1 infection steps in these cells. We will also test variations of our model that may allow for higher throughput studies as required for preclinical microbicide evaluation. In Aim 2, we will determine if infection of intraepithelial Langerhans and T cells constitutes a bottleneck favoring R5- over X4-tropic HIV-1 strains, therefore contributing to the predominance of R5 viruses during primary HIV infection. These studies will yield important information that will allow us to better understand mucosal HIV-1 transmission and selection, and will thus be useful for vaccine and microbicide design. Lay abstract: Worldwide, most HIV infections occur through the linings of the genital or intestinal tract following sexual contact. Information about the initial infection events at these sites is therefore needed to help in devising medical strategies for prevention, such as vaccines or locally delivered antiviral drugs. In this proposal, we will use a novel organ culture model of human vaginal tissue obtained from surgeries to characterize the entry pathways of HIV during the earliest stages of sexual transmission.